BACKGROUND AND PURPOSEAlthough the ongoing clinical trials of ABT-263 and ABT-199 in chronic lymphocytic leukaemia (CLL) have indicated that BH3 mimetics hold considerable promise, understanding the mechanism of CLL resistance to BH3 mimetics remains a challenge.
EXPERIMENTAL APPROACHThe LD 50 values of ABT-737, ABT-263 and ABT-199 in a number of primary CLL cells from 40 patients, were determined. The levels of Bcl-2 family proteins, including phosphorylated Bcl-2 (pBcl-2) and their interactions were measured by immunoblotting and co-immunoprecipitation. In vitro binding assays were performed by isothermal titration calorimetry and ELISA. BH3 profiling in isolated mitochondria was analysed.
KEY RESULTSThe ratio of (Mcl-1 + pBcl-2) to Bcl-2 expression provided the most significant predictive marker for the cytotoxic potential of ABT-737, ABT-263 and ABT-199 in the panel of CLL samples. Mechanistically, pBcl-2 inhibited the effects of the ABT compounds on the displacement of Bax and Bim from Bcl-2, thereby suppressing mitochondrial apoptosis. The ABT compounds exhibited 100-300-fold lower binding affinity to the glutamic acid, phosphomimetic, mutant of Bcl-2 (T69E, S70E and S87E; EEE-Bcl-2). BH3 peptides exhibited different rank orders of binding affinities to full-length WT-Bcl-2 and full-length EEE-Bcl-2.
CONCLUSIONS AND IMPLICATIONSOur study suggested that a structural alteration in the BH3-binding groove was induced by phosphorylation of Bcl-2. Our data also provided a framework to overcome resistance of CLL cells to the ABT compounds by combining pBcl-2 kinase inhibitors with the ABT compounds.Abbreviations CLL, chronic lymphocytic leukaemia; ITC, isothermal titration calorimetry; MOMP, mitochondrial outer membrane permeabilization BJP British Journal of Pharmacology
By means of limited proteolysis assay, three-dimensional NMR, X-ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl-2 homology 3 (BH3) domain of Mcl-1 has been identified as a conformational switch which controls Mcl-1 ubiquitination. Noxa binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl-1. In contrast, Bim binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl-1 inhibitor, which locates at the BH3 domain of Mcl-1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro-apoptotic partners, but also facilitates Mcl-1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.
Background and Purpose
The biological significance of the multi‐site phosphorylation of Bcl‐2 at its loop region (T69, S70 and S87) has remained controversial for decades. This is a major obstacle for understanding apoptosis and anti‐tumour drug development.
Experimental Approach
We established a mathematical model into which a phosphorylation and de‐phosphorylation process of Bcl‐2 was integrated. Paclitaxel‐treated breast cancer cells were used as experimental models. Changes in the kinetics of binding with its critical partners, induced by phosphorylation of Bcl‐2 were experimentally obtained by surface plasmon resonance, using a phosphorylation‐mimicking mutant EEE‐Bcl‐2 (T69E, S70E and S87E).
Key Results
Mathematical simulations combined with experimental validation showed that phosphorylation regulates Bcl‐2 with different dynamics depending on the extent of Bcl‐2 phosphorylation and the phosphorylated Bcl‐2‐induced changes in binding kinetics. In response to Bcl‐2 homology 3 (BH3)‐only protein Bmf stress, Bcl‐2 phosphorylation switched from diminishing to enhancing the Bcl‐2 anti‐apoptotic ability with increased phosphorylation of Bcl‐2, and the turning point was 50% Bcl‐2 phosphorylation induced by 0.2 μM paclitaxel treatment. In contrast, Bcl‐2 phosphorylation enhanced the anti‐apoptotic ability of Bcl‐2 towards other BH3‐only proteins Bim, Bad and Puma, throughout the entire phosphorylation procedure.
Conclusions and Implications
The model could accurately predict the effects of anti‐tumour drugs that involve the Bcl‐2 family pathway, as shown with ABT‐199 or etoposide.
By means of limited proteolysis assay, three‐dimensional NMR, X‐ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl‐2 homology 3 (BH3) domain of Mcl‐1 has been identified as a conformational switch which controls Mcl‐1 ubiquitination. NoxaBH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, BimBH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl‐1 inhibitor, which locates at the BH3 domain of Mcl‐1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro‐apoptotic partners, but also facilitates Mcl‐1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl‐1‐dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.
The design of a cross-acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two-face, two-turn α-helix is described. Docking studies and 2D (1)H, (15)N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl-1 and Bcl-2 proteins (Ki 0.079 and 0.056 μM, respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.
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